Sequence specific oligonucleotides are used extensively in the fields of molecular biology and probe diagnostics for such varied purposes as, for example, site directed mutagenesis, gene assembly, and as DNA probes to identify the presence of specific nucleic acid sequences in patient test samples. In addition, oligonucleotide probe mixtures have been prepared in research laboratories, based on the protein sequence of a resulting gene product and the degeneracy of the DNA code, to identify and characterize the nucleic acid sequences responsible for encoding these gene products. Even more recently, research has been directed toward the use of oligonucleotide sequences in a field which has become known as "antisense" therapy, whereby the oligonucleotides act as therapeutic agents to combat human diseases caused by vital and bacterial pathogens.
Oligonucleotide products have been generated in a number of different ways including both organic synthesis techniques and traditional cloning procedures. Organic synthesis techniques have been optimized to achieve moderate quantities of oligonucleotides. These synthetic methods include techniques referred to as the di-ester, the tri-ester, the H-phosphonate, and the phosphoramidite methodologies, with the solid support synthesis of oligonucleotides by the phosphoramidite method being the most widely employed. A review of current methods of oligonucleotide synthesis is provided in Caruthers, ch. 1, Oligonucleotides, Antisense Inhibitors of Gene Expression, ed., Cohen, CRC Press, Inc., Boca Rotan, Fla. (1989). Currently available technologies and instrumentation are, however, still limited with respect to the quantities of the synthetic oligonucleotide products that can be generated. In addition, the relatively harsh chemistries of these synthetic methods can produce a significant percentage of chemically altered oligonucleotides in the final product.
Because these chemical alterations represent relatively subtle changes, synthetic oligonucleotides are nevertheless suitable for many purposes. For example, the chemical alterations in synthetic oligonucleotides do not significantly interfere with hybridization, enabling these oligonucleotides to function effectively as DNA probes in most diagnostic applications. Similarly, in site directed mutagenesis, where entire colonies or plaques are derived from a single oligonucleotide incorporation, a biological screen is inherently provided for the desired mutation event.
However, certain applications require that a higher integrity product be employed to achieve optimal results. Chemical alteration typically becomes a problem where the oligonucleotides are used in an application which requires or induces recognition of the oligonucleotide by a biologically active substance, most typically an enzyme. For example, oligonucleotide integrity becomes an issue in gene assembly where polymerase and ligase recognition of the oligonucleotide is required for cloning. This problem is particularly acute in the assembly of longer genes, because the opportunity for error to arise from chemical alteration increases proportionately with the length of the gene.
The issue of oligonucleotide integrity is even more problematic in a therapeutic application where the therapeutic oligonucleotide must interact with a variety of biologically active components in the patient's body. Further, any degree of incomplete deprotection or chemical modification in the therapeutic oligonucleotide product might trigger an immune response, or worse, impart a cytotoxic effect to the patient. It is therefore questionable whether synthetic oligonucleotides can be effectively employed as therapeutic agents. This problem is further exacerbated by the fact that the exact nature of these subtle changes or errors are difficult, if not impossible, to characterize by current methods of chemical analysis. Mandecki et al, Biotechniques, 9(1), 56-59 (1990).
Traditional cloning techniques provide an alternative method for producing oligonucleotides. In cloning, the desired oligonucleotide sequence is inserted into an appropriate vector which is subsequently used to transform a host cell which, as it grows, amplifies the inserted oligonucleotide. The oligonucleotide can be synthesized in vitro and then inserted into a vector which is amplified by growth, as disclosed in U.S. Pat. No. 4,293,652. The inserted oligonucleotide sequence, if appropriately designed, can then be excised, for example by cutting with a restriction enzyme, and subsequently purified from extraneous plasmid DNA by standard techniques.
Oligonucleotides generated by cloning do not carry the danger of chemical modification. As a result, these oligonucleotide products appear as native, or "wild type", oligonucleotides which will, for example, be recognized by enzymes. Cloning procedures, however, do not tend to be economical or amenable to large scale production of oligonucleotides. Because the inserted oligonucleotide sequence represents only a small portion of the plasmid sequence, the weight yield of product DNA is very small compared with the weight yield of the plasmid. Even the insertion of tandem repeats of the desired oligonucleotide sequence is likely to be of no avail in improving yield, because biological host cell systems typically excise tandem repeats during growth.
Target amplification, a technique which has been employed to improve sensitivity in probe diagnostics, provides yet another, albeit "untraditional", type of oligonucleotide synthesis. For example, U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose a process known as polymerase chain reaction (PCR), wherein two oligonucleotide primers are employed such that the primers are complementary to the ends of different portions on opposite strands of a section of the target sequence. Following hybridization of these primers to the target, extension products complementary to the target sequence are formed in the presence of DNA polymerase and an excess of nucleoside triphosphates. The primers are oriented so that DNA synthesis by the polymerase proceeds across and through the region between the primers. The hybridized extension product is then denatured from the target and the cycle repeated, with extension product also acting as template for the formation of additional extension product in subsequent cycles of amplification. Each successive cycle theoretically doubles the amount of nucleic acid synthesized in the previous cycle, resulting in exponential accumulation of amplified product.
The PCR technology has been suggested for use as a method to achieve the production of large quantities of oligonucleotides, in European Patent Application No. 359,545. PCR production of oligonucleotides is, however, still limited with respect to the quantity of final oligonucleotide product which can be generated by this method, because a stoichiometric amount of each synthetic oligonucleotide primer must be incorporated into each PCR extension product. (In reality, a large excess of these primers is required to drive the kinetics of the PCR reaction forward.)
Another drawback, most typically in therapeutic applications, lies in the inability of PCR to completely eliminate the nucleic acid integrity problems of organic synthesis, because the synthetic primers in fact become a portion of the PCR-amplified oligonucleotide. As a result, the primer-derived portion of the product will contain the same errors that compromise the quality of synthetic oligonucleotides. For this reason, PCR amplification products cannot be of significantly higher quality than than that of the starting oligonucleotide primers. Additionally, the Thermus aquaticus (Taq) DNA polymerase enzyme which is typically used in PCR amplifications is believed to add one or more nucleotides during amplification onto the 3'-ends of the products beyond the oligonucleotide primers. Denney et al, Amplifications, A forum for PCR users, 4, 25-26 (March, 1990). It is also believed that Taq DNA polymerase can add bases onto the 3'-end of oligonucleotides in solution. Denney et al, ibid. This would result in a product which is not sequence specific.
Other types of target amplification which have been developed for diagnostic applications also warrant mention. International Patent Application No. 89/02649 discloses a type of amplification procedure generally referred to as ligase chain reaction (LCR), wherein presynthesized pairs of amplification probes which hybridize contiguously to a section of the target sequence are ligated to form the complementary amplification product. As with PCR, the completed amplification product is separated from the target by heat denaturation, and the process repeated with both the target and amplification product acting as a template in subsequent cycles. The primary advantage of the LCR method over prior organic synthesis methods lies in its ability to generate longer oligonucleotides from the shorter presynthesized amplification probes. The final oligonucleotide product can be synthetic, wild type, or a combination of both, depending upon the composition of the presynthesized probes.
Yet another type of diagnostic amplification method, referred to as catalytic hybridization amplification ("CHA"), is disclosed in International Patent Application No. 89/09284. In this method, multiple copies of the complement of a portion of a target sequence are "generated" from the target-catalyzed cleavage of longer presynthesized complementary probe sequences. Cleavage of the longer probes occurs only where the target hybridizes to one of an excess of these complementary detection probes in such a way that the target sequence catalyzes selective cleavage of only that complementary probe. Because the target remains intact, it can be recycled through the reaction, enabling it to react with more than one of the complementary detection probes, thus leading to a large amplification of signal through proliferation of the shorter cleaved pieces. CHA reactions, however, fail to provide an advantage in the synthesis of oligonucleotide products, because longer presynthesized oligonucleotides are simply being cleaved into smaller pieces.
It is an object of the present invention to provide a cost efficient method for producing large quantities of high quality oligonucleotides.
It is a further object of the present invention to provide a method for producing large quantities of oligonucleotides that can be useful in a diagnostic setting.